Mutant KRas inhibitors

ABSTRACT

Compositions and methods for inhibits the binding of GTP to oncogenic mutant KRas are disclosed. These compositions may be used in method to treat a subject with cancer. In particular, the compositions may be used to treat cancers involving overactive Ras signaling.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication 62/152,333, filed Apr. 24, 2015, which is herebyincorporated by reference herein in its entirety.

BACKGROUND

Ras activation is catalyzed by GEFs such as SOS that exchange bound GDPfor GTP, whereas Ras inactivation is catalyzed by GAPs leading to thehydrolysis of GTP to GDP. Oncogenic mutations in KRas impair GTPhydrolysis, leading to a constitutively activated, GTP-bound int KRas,which contributes significantly to human cancer pathogenesis and patienttumor resistance. The purpose of this research program is to identifysmall molecules that bind mt KRas directly and shift its state from GTPto GDP, inhibiting its ability to bind its effectors. Although thepicomolar affinity for GTP and the difficulty in identifying allostericsites have made it hard to target directly Ras, evidence for thefeasibility of targeting Ras has been reported. Recently, one reportprovided compelling evidence of small molecules that can bind covalentlyto Cys, occupy a pocket composed largely of the switch II region of G12Cmt KRas, and inhibit the interaction of Ras-GTP with its effectors.Other recent reports identified small molecules that inhibitedSOS-mediated nucleotide exchange by binding to a pocket adjacent to theswitch PH regions of KRas. Although compounds that exclusively inhibitGDP exchange for GTP may be effective in tumors that depend on wt Rasfor their malignancy, they are less likely to affect tumors with mtGTP-bound Ras.

SUMMARY

Compositions and methods for inhibiting the binding of GTP to oncogenicmutant KRas are disclosed. These compositions may be used in method totreat a subject with cancer. In particular, the compositions may be usedto treat cancers involving overactive Ras signaling.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A and FIG. 1B show optimization of the mant-GDP (FIG. 1A) and theGST-RBG (FIG. 1B) assays

FIG. 2 shows screening TPIMS scaffold ranking library identified hitsthat inhibited GTP from displacing want-GDP from mtG12DKras. The mostpotent 2239 mixture was de-convoluted through positional scanninglibraries.

FIG. 3A, FIG. 3B, and FIG. 3C show screening the TPIMS scaffold rankinglibrary and subsequent de-convolution through positional scanninglibraries led to the identification of 2239-87 which inhibits theability of GTP to displace mint-GDP from mt G12D.

FIG. 4 is a group of chemical structures of compounds as disclosedherein.

FIG. 5 is a group of chemical structures of compounds as disclosedherein.

FIG. 6 is a group of chemical structures of compounds with their percentinhibition.

DETAILED DESCRIPTION

The term “subject” refers to any individual who is the target ofadministration or treatment. The subject can be a vertebrate, forexample, a mammal. Thus, the subject can be a human or veterinarypatient. The term “patient” refers to a subject under the treatment of aclinician, e.g., physician.

A “pharmaceutically acceptable” component is one that is suitable foruse with humans and/or animals without undue adverse side effects (suchas toxicity, irritation, and allergic response) commensurate with areasonable benefit/risk ratio.

“Pharmaceutically acceptable salt” refers to a salt that ispharmaceutically acceptable and has the desired pharmacologicalproperties. Such salts include those that may be formed where acidicprotons present in the compounds are capable of reacting with inorganicor organic bases. Suitable inorganic salts include those formed with thealkali metals, e.g., sodium, potassium, magnesium, calcium, andaluminum. Suitable organic salts include those formed with organic basessuch as the amine bases, e.g., ethanolamine, diethanolamine,triethanolamine, tromethamine, N-methylglucamine, and the like. Suchsalts also include acid addition salts formed with inorganic acids(e.g., hydrochloric and hydrobromic acids) and organic acids (e.g.,acetic acid, citric acid, maleic acid, and the alkane- andarene-sulfonic acids such as methanesulfonic acid and benzenesulfonicacid). When two acidic groups are present, a pharmaceutically acceptablesalt may be a mono-acid-mono-salt or a di-salt; similarly, where thereare more than two acidic groups present, some or all of such groups canbe converted into salts.

“Pharmaceutically acceptable excipient” refers to an excipient that isconventionally useful in preparing a pharmaceutical composition that isgenerally safe, non-toxic, and desirable, and includes excipients thatare acceptable for veterinary use as well as for human pharmaceuticaluse. Such excipients can be solid, liquid, semisolid, or, in the case ofan aerosol composition, gaseous.

A “pharmaceutically acceptable carrier” is a carrier, such as a solvent,suspending agent or vehicle, for delivering the disclosed compounds tothe patient. The carrier can be liquid or solid and is selected with theplanned manner of administration in mind. Liposomes are also apharmaceutical carrier. As used herein, “carrier” includes any and allsolvents, dispersion media, vehicles, coatings, diluents, antibacterialand antifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated.

The term “therapeutically effective amount” as used herein means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician. In reference to cancers or other unwanted cellproliferation, an effective amount comprises an amount sufficient tocause a tumor to shrink and/or to decrease the growth rate of the tumor(such as to suppress tumor growth) or to prevent or delay other unwantedcell proliferation. In some embodiments, an effective amount is anamount sufficient to delay development. In some embodiments, aneffective amount is an amount sufficient to prevent or delay occurrenceand/or recurrence. An effective amount can be administered in one ormore doses. In the case of cancer, the effective amount of the drug orcomposition may: (i) reduce the number of cancer cells; (ii) reducetumor size; (iii) inhibit, retard, slow to some extent and preferablystop cancer cell infiltration into peripheral organs; (iv) inhibit(i.e., slow to some extent and preferably stop) tumor metastasis; (v)inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrenceof tumor; and/or (vii) relieve to some extent one or more of thesymptoms associated with the cancer.

Effective amounts of a compound or composition described herein fortreating a patient can include about 0.1 to about 1000 mg/Kg of bodyweight of the subject/day, such as from about 1 to about 100 mg/Kg/day,especially from about 10 to about 100 mg/Kg/day. The doses can be acuteor chronic. A broad range of disclosed composition dosages are believedto be both safe and effective.

The term “treatment” refers to the medical management of a patient withthe intent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

The term “inhibit” refers to a decrease in an activity, response,condition, disease, or other biological parameter. This can include butis not limited to the complete ablation of the activity, response,condition, or disease. This may also include, for example, a 10%reduction in the activity, response, condition, or disease as comparedto the native or control level. Thus, the reduction can be a 10, 20, 30,40, 50, 60, 70, 80, 90, 100% or any amount of reduction in between ascompared to native or control levels.

The term “cancer” or “malignant neoplasm” refers to a cell that displaysuncontrolled growth, invasion upon adjacent tissues, and oftenmetastasis to other locations of the body.

The cancer of the disclosed methods can be any cell in a subjectundergoing unregulated growth, invasion, or metastasis that involvesoveractive Ras signaling. In some aspects, the cancer can be anyneoplasm or tumor for which radiotherapy is currently used.Alternatively, the cancer can be a neoplasm or tumor that is notsufficiently sensitive to radiotherapy using standard methods. Thus, thecancer can be a sarcoma, lymphoma, leukemia, carcinoma, blastoma, orgerm cell tumor. A representative but non-limiting list of cancers thatthe disclosed compositions can be used to treat include lymphoma, B celllymphoma, T cell lymphoma, mycosis fungoi des, Hodgkin's Disease,myeloid leukemia, bladder cancer, brain cancer, nervous system cancer,head and neck cancer, squamous cell carcinoma of head and neck, kidneycancer, lung cancers such as small cell lung cancer and non-small celllung cancer, neuroblastoma/glioblastoma, ovarian cancer, pancreaticcancer, prostate cancer, skin cancer, liver cancer, melanoma, squamouscell carcinomas of the mouth, throat, larynx, and lung, colon cancer,cervical cancer, cervical carcinoma, breast cancer, epithelial cancer,renal cancer, genitourinary cancer, pulmonary cancer, esophagealcarcinoma, head and neck carcinoma, large bowel cancer, hematopoieticcancers; testicular cancer; colon and rectal cancers, prostatic cancer,and pancreatic cancer.

Chemical Definitions

Terms used herein will have their customary meaning in the art unlessspecified otherwise. The organic moieties mentioned when definingvariable positions within the general formulae described herein (e.g.,the term “halogen”) are collective terms for the individual substituentsencompassed by the organic moiety. The prefix C_(n)-C_(m) indicates ineach case the possible number of carbon atoms in the group.

The term “alkyl,” as used herein, refers to saturated straight,branched, primary, secondary or tertiary hydrocarbons, including thosehaving 1 to 20 atoms. In some examples, alkyl groups will includeC₁-C₁₂, C₁-C₁₀, C₁-C₈, C₁-C₆, C₁-C₅, C₁-C₄, C₁-C₃, or C₁-C₂ alkylgroups. Examples of C₁-C₁₀ alkyl groups include, but are not limited to,methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl,2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl,3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl,1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl,3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl,1,3-dimethytbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl,3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl,1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl,heptyl, octyl, 2-ethylhexyl, nonyl and decyl groups, as well as theirisomers. Examples of C₁-C₄-alkyl groups include, for example, methyl,ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, and1,1-dimethylethyl groups.

Cyclic alkyl groups or “cycloalkyl” groups include cycloalkyl groupshaving from 3 to 10 carbon atoms. Cycloalkyl groups can include a singlering, or multiple condensed rings. In some examples, cycloalkyl groupsinclude C₃-C₄, C₄-C₇, C₅-C₇, C₄-C₆, or C₅-C₆ cyclic alkyl groups.Non-limiting examples of cycloalkyl groups include adamantyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl and the like.

Alkyl and cycloalkyl groups can be unsubstituted or substituted with oneor more moieties chosen from alkyl, halo, haloalkyl, hydroxyl, carboxyl,acyl, acyloxy, amino, alkyl- or dialkylamino, amino, arylamino, alkoxy,aryloxy, nitro, cyano, azide, thiol, imino, sulfonic acid, sulfate,sulfonyl, sulfanyl, sulfinyl, sulfamonyl, ester, phosphoryl, phosphinyl,phosphoryl, phosphine, thioester, thioether, acid halide, anhydride,oxime, hydrazine, carbamate, phosphoric acid, phosphate, phosphonate, orany other viable functional group that does not inhibit the biologicalactivity of the compounds of the invention, either unprotected, orprotected as necessary, as known to those skilled in the art, forexample, as described in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Third Edition, 1999, hereby incorporatedby reference.

Terms including the term “alkyl,” such as “alkylamino” or“dialkylamino,” will be understood to comprise an alkyl group as definedabove linked to another functional group, where the group is linked tothe compound through the last group listed, as understood by those ofskill in the art.

The term “alkenyl,” as used herein, refers to both straight and branchedcarbon chains which have at least one carbon-carbon double bond. In someexamples, alkenyl groups can include C₂-C₂₀ alkenyl groups. In otherexamples, alkenyl can include C₂-C₁₂, C₂-C₁₀, C₂-C₈, C₂-C₆, or C₂-C₄alkenyl groups. In one embodiment of alkenyl, the number of double bondsis 1-3, in another embodiment of alkenyl, the number of double bonds isone or two. Other ranges of carbon-carbon double bonds and carbonnumbers are also contemplated depending on the location of the alkenylmoiety on the molecule. “C₂-C₁₀-alkenyl” groups can include more thanone double bond in the chain. The one or more unsaturations within thealkenyl group can be located at any position(s) within the carbon chainas valence permits. In some examples, when the alkenyl group iscovalently bound to one or more additional moieties, the carbon atom(s)in the alkenyl group that are covalently bound to the one or moreadditional moieties are not part of a carbon-carbon double bond withinthe alkenyl group. Examples of alkenyl groups include, but are notlimited to, ethenyl, 1-propenyl, 2-propenyl, 1-methyl-ethenyl,1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl,2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl;1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl,2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl,2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl,2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl,1,2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl,1-ethyl-2-propenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl,3-methyl-1-pentenyl, 4-methyl-1-pentenyl, 1-methyl-2-pentenyl,2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl,1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl,4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl,3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl,1,1-dimethyl-3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethyl-2-butenyl,1,2-dimethyl-3-butenyl, 1,3-dimethyl-1-butenyl, 1,3-dimethyl-2-butenyl,1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-1-butenyl,2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-1-butenyl,3,3-dimethyl-2-butenyl, 1-ethyl-1-butenyl, 1-ethyl-2-butenyl,1-ethyl-3-butenyl, 2-ethyl-1-butenyl, 2-ethyl-2-butenyl,2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl,1-ethyl-1-methyl-2-propenyl, 1-ethyl-2-methyl-1-propenyl and1-ethyl-2-methyl-2-propenyl groups.

The term “alkynyl,” as used herein, refers to both straight and branchedcarbon chains which have at least one carbon-carbon triple bond. In oneembodiment of alkynyl, the number of triple bonds is 1-3; in anotherembodiment of alkynyl, the number of triple bonds is one or two. In someexamples, alkynyl groups include from C₂-C₂₀ alkynyl groups. In otherexamples, alkynyl groups can include C₂-C₁₂, C₂-C₁₀, C₂-C₈, C₂-C₆ orC₂-C₄ alkynyl groups. Other ranges of carbon-carbon triple bonds andcarbon numbers are also contemplated depending on the location of thealkenyl moiety on the molecule. For example, the term “C₂-C₁₀-alkynyl”as used herein refers to a straight-chain or branched unsaturatedhydrocarbon group having 2 to 10 carbon atoms and containing at leastone triple bond, such as ethynyl, prop-1-yn-1-yl, prop-2-yn-1-yl,n-but-1-yn-1-yl, n-but-1-yn-3-yl, n-but-1-yn-4-yl, n-but-2-yn-1-yl,n-pent-1-yn-1-yl, n-pent-1-yn-3-yl, n-pent-1-yn-4-yl, n-pent-1-yn-5-yl,n-pent-2-yn-1-yl, n-pent-2-yn-5-yl, 3-methylbut-1-yn-3-yl,3-methylbut-1-yn-4-yl, n-hex-1-yn-1-yl, n-hex-1-yn-3-yl,n-hex-1-yn-4-yl, n-hex-1-yn-5-yl, n-hex-2-yn-1-yl, n-hex-2-yn-4-yl,n-hex-2-yn-5-yl, n-hex-2-yn-6-yl, n-hex-3-yn-1-yl, n-hex-3-yn-2-yl,3-methylpent-1-yn-1-yl, 3-methylpent-1-yn-3-yl, 3-methylpent-1-yn-4-yl,3-methylpent-1-yn-5-yl, 4-methylpent-1-yn-1-yl, 4-methylpent-2-yn-4-yl,and 4-methylpent-2-yn-5-yl groups.

Alkenyl and alkynyl groups can be unsubstituted or substituted with oneor more moieties chosen from alkyl, halo, haloalkyl, hydroxyl, carboxyl,acyl, acyloxy, amino, alkyl- or dialkylamino, amido, arylamino, alkoxy,aryloxy, nitro, cyano, azido, thiol, imino, sulfonic acid, sulfate,sulfonyl, sulfanyl, sulfinyl, sulfamoyl, ester, phosphonyl, phosphinyl,phosphoryl, phosphine, thioester, thioether, acid halide, anhydride,oxime, hydrazine, carbamate, phosphoric acid, phosphate, phosphonate, orany other viable functional group that does not inhibit the biologicalactivity of the compounds of the invention, either unprotected, orprotected as necessary, as known to those skilled in the art, forexample, as described in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Third Edition, 1999.

The term “aryl,” as used herein, refers to a monovalent aromaticcarbocyclic group of from 6 to 14 carbon atoms. Aryl groups can includea single ring or multiple condensed rings. In some examples, aryl groupsinclude C₆-C₁₀ aryl groups. Aryl groups include, but are not limited to,phenyl, biphenyl, naphthyl, tetrahydronaphtyl, phenylcyclopropyl andindanyl. Aryl groups can be unsubstituted or substituted by one or moremoieties chosen from halo, cyano, nitro, hydroxy, mercapto, amino,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, halloalkyl,haloallkenyl, haloalkynyl, halocycloalkyl, halocycloalkenyl, alkoxy,alkenyloxy, alkynyloxy, haloalkoxy, haloalkenyloxy, haloalkynyloxy,cycloalkoxy, cycloalkenyloxy, halocycloalkoxy, halocycloalkenyloxy,alkylthio, haloalkylthio, cycloalkylthio, halocycloalkylthio,alkylsulfinyl, alkenylsulfinyl, alkynyl-sulfonyl, haloalkylsulfinyl,haloalkenylsulfinyl, haloalkynylsulfinyl, alkylsulfonyl,alkenylsulfonyl, alkynylsulfonyl, haloalkyl-sulfonyl,haloalkenylsulfonyl, haloalkynylsulfonyl, alkylamino, alkenylamino,alkynylatnino, di(alkyl)amino, di(alkenyl)-amino, di(alkynyl)amino, ortrialkylsilyl.

The term “alkylaryl,” as used herein, refers to an aryl group that isbonded to a parent compound through a diradical alkylene bridge,(—CH₂—)_(n), where n is 1-12 (e.g., n is from 1 to 6) and where “aryl”is as defined above. The term “arylalkyl,” as used herein, refers to anaryl group, as defined above, which is substituted by an alkyl group, asdefined above.

The term “alkylcycloalkyl,” as used herein, refers to a cycloalkyl groupthat is bonded to a parent compound through a diradical alkylene bridge,(—CH₂—)_(n), where n is 1-12 n is from 1 to 6) and where “cycloalkyl” isas defined above.

The term “alkoxy,” as used herein, refers to alkyl-O—, wherein alkylrefers to an alkyl group, as defined above. Similarly, the terms“alkenyloxy” “alkynyloxy,” and “cycloalkoxy,” refer to the groupsalkenyl-O—, alkynyl-O—, and cycloalkyl-O—, respectively, whereinalkenyl, alkynyl, and cycloalkyl are as defined above. Examples ofC₁-C₆-alkoxy groups include, but are not limited to, methoxy, ethoxy,C₂H₅—CH₂O—, (CH₃)₂CHO—, n-butoxy, C₂H₅—CH(CH₃)O—, (CH₃)₂CH—CH₂O—,(CH₃)₃CO—, n-pentoxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy,1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethyl-propoxy,1-ethylpropoxy, n-hexoxy, 1-methylpentoxy, 2-methylpentoxy,3-methylpentoxy, 4-methylpentoxy, 1,1-dimethylbutoxy,1,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2,2-dimethylbutoxy,2,3-dimethylbutoxy, 3,3-dimethylbutoxy, 1-ethylbutoxy, 2-ethylbutoxy,1,1,2-trimethylpropoxy, 1,2,2-trimethylpropoxy,1-ethyl-1-tnethylpropoxy, and 2-ethyl-2-methylpropoxy.

The term “alkylthio,” as used herein, refers to alkyl-S—, wherein alkylrefers to an alkyl group, as defined above. Similarly, the term“cycloalkylthio,” refers to cycloalkyl-S— where cycloalkyl are asdefined above.

The term “alkylsulfinyl,” as used herein, refers to alkyl-S(O)—, whereinalkyl refers to an alkyl group, as defined above.

The term “alkylsulfonyl,” as used herein, refers to alkyl-S(O)₂—,wherein alkyl is as defined above.

The terms “alkylamino” and “dialkylamino,” as used herein, refer toalkyl-NH— and (alkyl)₂N— groups, where alkyl is as defined above.

The terms “alkylcarbonyl,” “alkoxycarbonyl,” “alkylarninocarbonyl,” and“dialkylaminocarbonyl,” as used herein, refer to alkyl-C(O)—,alkoxy-C(O)—, alkylamino-C(O)— and dialkylamino-C(O)— respectively,where alkyl, alkoxy, alkylamino, and dialkylamino are as defined above.

The term “heteroaryl,” as used herein, refers to a monovalent aromaticgroup of from 1 to 15 carbon atoms (e.g., from 1 to 10 carbon atoms,from 2 to 8 carbon atoms, from 3 to 6 carbon atoms, or from 4 to 6carbon atoms) having one or more heteroatoms within the ring. Theheteroaryl group can include from 1 to 4 heteroatoms, from 1 to 3heteroatoms, or from 1 to 2 heteroatoms. In some examples, theheteroatom(s) incorporated into the ring are oxygen, nitrogen, sulfur,or combinations thereof. When present, the nitrogen and sulfurheteroatoms can optionally be oxidized. Heteroaryl groups can have asingle ring (e.g., pyridyl or furyl) or multiple condensed ringsprovided that the point of attachment is through a heteroaryl ring atom.Preferred heteroaryls include pyridyl, piridazinyl, pyrimidinyl,pyrazinyl, triazinyl, pyrrolyl, indolyl, quinolinyl, isoquinolinyl,quinazolinyl, quinoxalinnyl, furanyl, thiophenyl, furyl, pyrrolyl,imidazolyl, oxazolyl, isoxazolyl, isothiazolyl, pyrazolyl benzofuranyl,and benzothiophenyl. Heteroaryl rings can be unsubstituted orsubstituted by one or more moieties as described for aryl above.

The term “alkylheteroaryl,” as used herein, refers to a heteroaryl groupthat is bonded to a parent compound through a diradical alkylene bridge,(—CH₂—)_(n), where n is 1-12 and where “heteroaryl” is as defined above.

The terms “heterocyclyl,” “heterocyclic” and “heterocyclo” are usedherein interchangeably, and refer to fully saturated or unsaturated,cyclic groups, for example, 3 to 7 membered monocyclic or 4 to 7membered monocyclic; 7 to 11 membered bicyclic, or 10 to 15 memberedtricyclic ring systems, having one or more heteroatoms within the ring.The heterocyclyl group can include from 1 to 4 heteroatoms, from 1 to 3heteroatoms, or from 1 to 2 heteroatoms. In some examples, theheteroatom(s) incorporated into the ring are oxygen, nitrogen, sulfur,or combinations thereof. When present, the nitrogen and sulfurheteroatoms can optionally be oxidized, and the nitrogen heteroatoms canoptionally be quaternized. The heterocyclyl group can be attached at anyheteroatom or carbon atom of the ring or ring system and can beunsubstituted or substituted by one or more moieties as described foraryl groups above.

Exemplary monocyclic heterocyclic groups include, but are not limitedto, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl,imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl,isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl,piperidinyi, piperazinyl, 2-oxopiperazinyl. 2-oxopiperidinyl,2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, 4-piperidonyl, pyridinyl,pyrazinyl, pyrimidinyl, pyridazinyl, tetrahydropyranyl, morpholinyl,thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone,1,3-dioxolane and tetrahydro-1,1-dioxothienyl, triazolyl, triazinyl, andthe like.

Exemplary bicyclic heterocyclic groups include, but are not limited to,indolyl, benzothiazolyl, benzoxazolyl, benzodioxolyl, benzothienyl,quinuclidinyl, quinolinyl, tetra-hydroisoquinolinyl, isoquinolinyl,benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, chromonyl,coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl,pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl,furo[3,2-b]pyridinyl] or furo[2,3-b]pyridinyl), dihydroisoindolyl,dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl),tetrahydroquinolinyl and the like.

Exemplary tricyclic heterocyclic groups include carbazolyl, benzidolyl,phenanthrolinyl, acridinyl, phenanthridinyl, xanthenyl, and the like.

The term “alkylheterocyclyl,” as used herein, refers to a heterocyclylgroup that is bonded to a parent compound through a diradical alkylenebridge, (—CH₂—)_(n), where n is 1-12 and where “heterocyclyl” is asdefined above. The term “heterocyclylalkyl,” as used herein, refers to aheterocyclyl group, as defined above, which is substituted by an alkylgroup, as defined above.

Heretrocyclyl and heteroaryl groups can be unsubstituted or substitutedwith one or more moieties chosen from alkyl, halo, haloalkyl, hydroxyl,carboxyl, acyl, acyloxy, amino, alkyl- or dialkylamino, amido,arylamino, alkoxy, aryloxy, nitro, cyano, azido, thiol, imino, sulfonicacid, sulfate, sulfonyl, sulfanyl, sulfinyl, sulfamonyl, ester,phosphonyl, phosphinyl, phosphoryl, phosphine, thioester, thioether,acid halide, anhydride, oxime, hydrazine, carbamate, phosphoric acid,phosphate, phosphonate, or any other viable functional group that doesnot inhibit the biological activity of the compounds of the invention,either unprotected, or protected as necessary, as known to those skilledin the art, for example, as described in Greene, et al., ProtectiveGroups in Organic Synthesis, John Wiley and Sons, Third Edition, 1999

The term “halogen,” as used herein, refers to the atoms fluorine,chlorine, bromine and iodine. The prefix halo- (e.g., as illustrated bythe term haloalkyl) refers to all degrees of halogen substitution, froma single substitution to a perhalo substitution (e.g., as illustratedwith methyl as chloromethyl (—CH₂Cl), dichloromethyl (—CHCl₂),trichloromethyl (—CCl₃)).

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer, diastereomer, and meso compound,and a mixture of isomers, such as a racemic or scalemic mixture.

Reference will now be made in detail to specific aspects of thedisclosed materials, compounds, compositions, articles, and methods,examples of which are illustrated in the accompanying Examples andFigures.

Compositions and Methods

Compositions and methods for inhibits the binding of GTP to oncogenicmutant KRas are disclosed. These compositions may be used in method totreat a subject with cancer. In particular, the compositions may be usedto treat cancers involving overactive Ras signaling.

In some embodiments, methods can involve administering an effectiveamount of a compound defined by Formula I below

wherein

-   -   R¹ is H, F, Cl, Br, CF₃, or an alkyl group or alkenyl group,        optionally substituted with amino, amido, alkyl, alkenyl,        alkynyl, alkoxyl, aryl, carbonyl, carboxylate, carbamyl, cyano,        ester, guanidinyl, halogen, haloalkyl, heteroaryl, hydroxyl,        nitrile, nitro, sulfinyl, sulfanyl, thiol, ureylene, or ureido;    -   R² is an alkyl group, an alkenyl group, an alkynyl group, a        cycloalkyl group, a heterocyclyl group, an aryl group, a        heteroaryl group, optionally substituted with amino, amino,        alkyl, alkenyl, alkynyl, alkoxyl, aryl, carbonyl, carboxylate,        carbamyl, cyano, ester, guanidinyl, halogen, haloalkyl,        heteroaryl, hydroxyl, nitrile, nitro, sulfinyl, sulfanyl, thiol,        ureylene, or ureido, or —CH₂—R⁴;    -   R³ is H, F, Cl, Br, CF₃, an alkyl group, or an alkenyl group;    -   R⁴ is an aryl group, an alkylaryl group, an arylalkyl group, a        cycloalkyl group, an alkylcycloalkyl group, a heterocyclyl        group, an alkylheterocyclyl group, a heteroaryl group, an        alkylheteroaryl group, an alkoxy group, or an alkylthio group        optionally substituted with amino, amido, alkyl, alkenyl,        alkynyl, alkoxyl, aryl, carbonyl, carboxylate, carbamyl, cyano,        ester, guanidinyl, halogen, haloalkyl, heteroaryl, hydroxyl,        nitrile, nitro, sulfinyl, sulfanyl, thiol, ureylene, or ureido;

or a pharmaceutical acceptable salt thereof, and optionally apharmaceutically acceptable carrier.

In specific examples, the disclosed compounds have R³ as alkyl, e.g.,C₁-8 alkyl, more specifically, C1-4 alkyl, more specifically methyl.

In specific examples, the disclosed compounds can have R2 asunsubstituted phenyl or phenyl substituted

In certain aspects, the disclosed compounds can have Formula IA, whichis a subgenus of Formula I where R³ is methyl and R² is phenyl.

wherein R¹ is as defined above. In specific examples, R¹ can beunsubstituted C₁₋₄ alkyl, for example isopropyl, or C₁₋₄ alkylsubstituted with hydroxyl, guanidinyl, phenyl (i.e., benzyl), phenol,tolyl, or heteroaryl.

In other aspects, the disclosed compounds can have Formula IB, which isa subgenus of Formula I where R³ is hydrogen and R² is substituted orunsubstituted phenyl and R¹ is as defined above.

wherein R⁵ is hydrogen amino, amido, alkyl, alkenyl, alkynyl, alkoxyl,aryl, carbonyl, carboxylate, carbamyl, cyano, ester, guanidinyl,halogen, haloalkyl, heteroaryl, hydroxyl, nitrile, nitro, sulfinyl,sulfanyl, thiol, ureylene, or ureido, or —CH₂—R⁴. In specific examples,R¹ can be unsubstituted alkyl, for example isopropyl, or C₁₋₄ alkylsubstituted with hydroxyl, guanidinyl, phenyl (i.e., benzyl), phenol,tolyl, or heteroaryl.

In certain embodiments, the compound can be

In certain embodiments, the compound can be

Other specific examples of compounds of Formulas I, IA, and IBA areshown in FIG. 4, FIG. 5, and FIG. 6, Pharmaceutically acceptable saltsof these compounds are also contemplated herein as are such compoundswith a pharmaceutically acceptable carrier.

Also provided are pharmaceutical compositions comprising atherapeutically effective amount of one or more of these compounds and apharmaceutically acceptable excipient.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

Methods or Use

Further provided herein are methods of treating or preventing cancer ina subject, comprising administering to the subject an effective amountof a compound or composition as disclosed herein. Additionally, themethod can further comprise administering an effective amount ofionizing radiation to the subject and/or another anti-cancer compound.Also disclosed are methods of imaging a cancer cell comprisingcontacting the cell with a compound as disclosed herein.

Methods of killing a tumor cell are also provided herein. The methodscomprise contacting a tumor cell with an effective amount of a compoundor composition as disclosed herein. The methods can further includeadministering a second compound or composition (e.g., an anticanceragent) or administering an effective amount of ionizing radiation to thesubject.

Also provided herein are methods of radiotherapy of tumors, comprisingcontacting the tumor with an effective amount of a compound orcomposition as disclosed herein and irradiating the tumor with aneffective amount of ionizing radiation. Methods of treating inflammationin a subject are further provided herein, the methods comprisingadministering to the subject an effective amount of a compound orcomposition as described herein. Optionally, the methods can furtherinclude administering a second compound or composition (e.g., ananti-inflammatory agent).

The disclosed subject matter also concerns methods for treating asubject having an oncological disorder or condition. In one embodiment,an effective amount of one or more compounds or compositions disclosedherein is administered to a subject having an oncological disorder andwho is in need of treatment thereof. The disclosed methods canoptionally include identifying a subject who is or can be in need oftreatment of an oncological disorder. The subject can be a human orother mammal, such as a primate (monkey, chimpanzee, ape, etc.), dog,cat, cow, pig, or horse, or other animals having an oncologicaldisorder. Means for administering and formulating compounds foradministration to a subject are known in the art, examples of which aredescribed herein. Oncological disorders include, but are not limited to,cancer and/or tumors of the anus, bile duct, bladder, bone, bone marrow,bowel (including colon and rectum), breast, eye, gall bladder, kidney,mouth, larynx, esophagus, stomach, testis, cervix, head, neck, ovary,lung, mesothelioma, neuroendocrine, penis, skin, spinal cord, thyroid,vagina, vulva, uterus, liver, muscle, pancreas, prostate, blood cells(including lymphocytes and other immune system cells), and brain.Specific cancers contemplated for treatment include carcinomas,Karposi's sarcoma, melanoma, mesothelioma, soft tissue sarcoma,pancreatic cancer, lung cancer, leukemia (acute lymphoblastic, acutemyeloid, chronic lymphocytic, chronic myeloid, and other), and lymphoma(Hodgkin's and non-Hodgkin's), and multiple myeloma.

Other examples of cancers that can be treated according to the methodsdisclosed herein are adrenocortical carcinoma, adrenocortical carcinoma,cerebellar astrocytoma, basal cell carcinoma, bile duct cancer, bladdercancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma,carcinoid tumor, central nervous system lymphoma, cervical cancer,chronic myeloproliferative disorders, colon cancer, cutaneous T-celllymphoma, endometrial cancer, ependymoma, esophageal cancer, gallbladdercancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, germcell tumor, glioma hairy cell leukemia, head and neck cancer,hepatocellular (liver) cancer, hypopharyngeal cancer, hypothalamic andvisual pathway glioma, intraocular melanoma, retinoblastoma, islet cellcarcinoma (endocrine pancreas), laryngeal cancer, lip and oral cavitycancer, liver cancer, medulloblastoma, Merkel cell carcinoma, squamousneck cancer with occult mycosis fungoides, myelodysplastic syndromes,myelogenous leukemia, nasal cavity and paranasal sinus cancer,nasopharyngeal cancer, neuroblastoma, non-small cell lungcancer, oralcancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreaticcancer, paranasal sinus and nasal cavity cancer, parathyroid cancer,penile cancer, pheochromocytoma, pineoblastoma and supratentorialprimitive neuroectoderrnal tumor, pituitary tumor, plasma cellneoplasm/multiple myeloma, pleuropulmonary blastoma, prostate cancer,rectal cancer, renal cell (kidney) cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, Ewing's sarcoma, soft tissuesarcoma, Sezary syndrome, skin cancer, small cell lung cancer, smallintestine cancer, supratentorial primitive neuroectodermal tumors,testicular cancer, thymic carcinoma, thymoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter, trophoblastictumor, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer,Waldenstrom's macroglobulinemia, and Wilms' tumor. In a preferredaspect, the cancer is pancreatic cancer.

Formulations and Methods of Administration

In vivo application of the disclosed compounds, and compositionscontaining them, can be accomplished by any suitable method andtechnique presently or prospectively known to those skilled in the art.For example, the disclosed compounds can be formulated in aphysiologically- or pharmaceutically-acceptable form and administered byany suitable route known in the art including, for example, oral, nasal,rectal, topical, and parenteral routes of administration. As usedherein, the term parenteral includes subcutaneous, intradermal,intravenous, intramuscular, intraperitoneal, and intrasternaladministration, such as by injection. Administration of the disclosedcompounds or compositions can be a single administration, or atcontinuous or distinct intervals as can be readily determined by aperson skilled in the art.

The compounds disclosed herein, and compositions comprising them, canalso be administered utilizing liposome technology, slow releasecapsules, implantable pumps, and biodegradable containers. Thesedelivery methods can, advantageously, provide a uniform dosage over anextended period of time. The compounds can also be administered in theirsalt derivative forms or crystalline forms.

The compounds disclosed herein can be formulated according to knownmethods for preparing pharmaceutically acceptable compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin (1995)describes formulations that can be used in connection with the disclosedmethods. In general, the compounds disclosed herein can be formulatedsuch that an effective amount of the compound is combined with asuitable carrier in order to facilitate effective administration of thecompound. The compositions used can also be in a variety of forms. Theseinclude, for example, solid, semi-solid, and liquid dosage forms, suchas tablets, pills, powders, liquid solutions or suspension,suppositories, injectable and infusible solutions, and sprays. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions also preferably includeconventional pharmaceutically-acceptable carriers and diluents which areknown to those skilled in the art. Examples of carriers or diluents foruse with the compounds include ethanol, dimethyl sulfoxide, glycerol,alumina, starch, saline, and equivalent carriers and diluents. Toprovide for the administration of such dosages for the desiredtherapeutic treatment, compositions disclosed herein can advantageouslycomprise between about 0.1% and 99%, and especially, 1 and 15% by weightof the total of one or more of the subject compounds based on the weightof the total composition including carrier or diluent.

Formulations suitable for administration include, for example, aqueoussterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions, which can include suspending agents and thickening agents.The formulations can be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and can be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions can be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, thecompositions disclosed herein can include other agents conventional inthe art having regard to the type of formulation in question.

Compounds disclosed herein, and compositions comprising them, can bedelivered to a cell either through direct contact with the cell or via acarrier means. Carrier means for delivering compounds and compositionsto cells are known in the art and include, for example, encapsulatingthe composition in a liposome moiety. Another means for delivery ofcompounds and compositions disclosed herein to a cell comprisesattaching the compounds to a protein or nucleic acid that is targetedfor delivery to the target cell. U.S. Pat. No. 6,960,648 and U.S.Application Publication Nos. 20030032594 and 20020120100 disclose aminoacid sequences that can be coupled to another composition and thatallows the composition to be translocated across biological membranes.U.S. Application Publiation No. 20020035243 also describes compositionsfor transporting biological moieties across cell membranes forintracellular delivery. Compounds can also be incorporated intopolymers, examples of which include poly (D-L lactide-co-glycolide)polymer for intracranial tumors; poly[bis(p-carboxyphenoxy)propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL);chondroitin; chitin; and chitosan.

For the treatment of oncological disorders, the compounds disclosedherein can be administered to a patient in need of treatment incombination with other antitumor or anticancer substances and/or withradiation and/or photodynamic therapy and/or with surgical treatment toremove a tumor. These other substances or treatments can be given at thesame as or at different times from the compounds disclosed herein. Forexample, the compounds disclosed herein can be used in combination withmitotic inhibitors such as taxol or vinblastine, alkylating agents suchas cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracilor hydroxyurea, DNA intercalators such as adriamycin or bleomycin,topoisomerase inhibitors such as etoposide or camptothecin,antiangiogenic agents such as angiostatin, antiestrogens such astamoxifen, and/or other anti-cancer drugs or antibodies, such as, forexample, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN(Genentech, Inc.), respectively, or an immunotherapeutic such asipilimumab and bortezomib.

In certain examples, compounds and compositions disclosed herein can belocally administered at one or more anatomical sites, such as sites ofunwanted cell growth (such as a tumor site or benign skin growth, e.g.,injected or topically applied to the tumor or skin growth), optionallyin combination with a pharmaceutically acceptable carrier such as aninert diluent. Compounds and compositions disclosed herein can besystemically administered, such as intravenously or orally, optionallyin combination with a pharmaceutically acceptable carrier such as aninert diluent, or an assimilable edible carrier for oral delivery. Theycan be enclosed in hard or soft shell gelatin capsules, can becompressed into tablets, or can be incorporated directly with the foodof the patient's diet. For oral therapeutic administration, the activecompound can be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, aerosol sprays, and the like.

The tablets, troches, pills, capsules, and the like can also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring can be added. Whenthe unit dosage form is a capsule, it can contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials can be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules can be coatedwith gelatin, wax, shellac, or sugar and the like. A syrup or elixir cancontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound canbe incorporated into sustained-release preparations and devices.

Compounds and compositions disclosed herein, including pharmaceuticallyacceptable salts, or hydrates thereof, can be administeredintravenously, intramuscularly, or intraperitoneally by infusion orinjection. Solutions of the active agent or its salts can be prepared inwater, optionally mixed with a nontoxic surfactant. Dispersions can alsobe prepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient, which are adapted for theextemporaneous preparation of sterile injectable or infitsible solutionsor dispersions, optionally encapsulated in liposomes. The ultimatedosage form should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. Optionally, the prevention of the action of microorganismscan be brought about by various other antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by e inclusion of agents that delay absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating a compoundand/or agent disclosed herein in the required amount in the appropriatesolvent with various other ingredients enumerated above, as required,followed by filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, compounds and agents disclosed herein can beapplied in as a liquid or solid. However, it will generally be desirableto administer them topically to the skin as compositions, in combinationwith a dermatologically acceptable carrier, which can be a solid or aliquid. Compounds and agents and compositions disclosed herein can beapplied topically to a subject's skin to reduce the size (and caninclude complete removal) of malignant or benign growths, or to treat aninfection site. Compounds and agents disclosed herein can be applieddirectly to the growth or infection site. Preferably, the compounds andagents are applied to the growth or infection site in a formulation suchas an ointment, cream, lotion, solution, tincture, or the like.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers, for example.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Useful dosages of the compounds and agents and pharmaceuticalcompositions disclosed herein can be determined by comparing their invitro activity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art.

Also disclosed are pharmaceutical compositions that comprise a compounddisclosed herein in combination with a pharmaceutically acceptablecarrier. Pharmaceutical compositions adapted for oral, topical orparenteral administration, comprising an amount of a compound constitutea preferred aspect. The dose administered to a patient, particularly ahuman, should be sufficient to achieve a therapeutic response in thepatient over a reasonable time frame, without lethal toxicity, andpreferably causing no more than an acceptable level of side effects ormorbidity. One skilled in the art will recognize that dosage will dependupon a variety of factors including the condition (health) of thesubject, the body weight of the subject, kind of concurrent treatment,if any, frequency of treatment, therapeutic ratio, as well as theseverity and stage of the pathological condition.

Kits

The disclosed subject matter also concerns a packaged dosage formulationcomprising in one or more containers at least one inhibitor compound orcomposition disclosed herein, e.g., any compound of Formulas I. Apackaged dosage formulation can optionally comprise in one or morecontainers a pharmaceutically acceptable carrier or diluent. A packageddosage formulation can also optionally comprise, in addition to aninhibitor compound or composition disclosed herein, other GTPinhibitors, or an immunotherapeutic such as ipilimumab.

Depending upon the disorder or disease condition to be treated, asuitable dose(s) can be that amount that will reduce proliferation orgrowth of the target cell(s). In the context of cancer, a suitabledose(s) is that which will result in a concentration of the active agentin cancer tissue, such as a malignant tumor, which is known to achievethe desired response. The preferred dosage is the amount which resultsin maximum inhibition of cancer cell growth, without unmanageable sideeffects. Administration of a compound and/or agent can be continuous orat distinct intervals, as can be determined by a person of ordinaryskill in the art.

To provide for the administration of such dosages for the desiredtherapeutic treatment, in some embodiments, pharmaceutical compositionsdisclosed herein can comprise between about 0.1% and 45%, andespecially, 1 and 15%, by weight of the total of one or more of thecompounds based on the weight of the total composition including carrieror diluents. Illustratively, dosage levels of the administered activeingredients can be: intravenous, 0.01 to about 20 mg/kg;intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous, 0.01 to about100 mg/kg; intramuscular, 0.01 to about 100 mg/kg; orally 0.01 to about200 mg/kg, and preferably about 1 to 100 mg/kg; intranasal instillation,0.01 to about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal(body) weight.

Also disclosed are kits that comprise a composition comprising acompound disclosed herein in one or more containers. The disclosed kitscan optionally include pharmaceutically acceptable carriers and/ordiluents. In one embodiment, a kit includes one or more othercomponents, adjuncts, or adjuvants as described herein. In anotherembodiment, a kit includes one or more anti-cancer agents, such as thoseagents described herein. In one embodiment, a kit includes instructionsor packaging materials that describe how to administer a compound orcomposition of the kit. Containers of the kit can be of any suitablematerial, e.g., glass, plastic, metal, etc., and of any suitable size,shape, or configuration. In one embodiment, a compound and/or agentdisclosed herein is provided in the kit as a solid, such as a tablet,pill, or powder form. In another embodiment, a compound and/or agentdisclosed herein is provided in the kit as a liquid or solution. In oneembodiment, the kit comprises an ampoule or syringe containing acompound and/or agent disclosed herein in liquid or solution form.

EXAMPLES

The following examples are set forth below to illustrate the methods,compositions, and results according to the disclosed subject matter.These examples are not intended to be inclusive of all aspects of thesubject matter disclosed herein, but rather to illustrate representativemethods, compositions, and results. These examples are not intended toexclude equivalents and variations of the present invention, which areapparent to one skilled in the art.

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric. There are numerous variations and combinations ofreaction conditions, e.g., component concentrations, temperatures,pressures, and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

EXAMPLE 1

Ras activation is catalyzed by GEFs such as SOS that exchange bound GDPfor GTP, whereas Ras inactivation is catalyzed by GAPS leading to thehydrolysis of GTP to GDP. Oncogenic mutations in KRas impair GTPhydrolysis, leading to a constitutively activated, GTP-bound mt KRas,which contributes significantly to human cancer pathogenesis and patienttumor resistance. The purpose of this example is to identify smallmolecules that bind mt KRas directly and shift its state from GTP toGDP, inhibiting its ability to bind its effectors. Although thepicomolar affinity for GTP and the difficulty in identifying allostericsites have made it hard to target directly Ras, evidence for thefeasibility of targeting Ras has been reported. Recently, one reportprovided compelling evidence of small molecules that can bind covalentlyto Cys, occupy a pocket composed largely of the switch II region of G12Cmt KRas, and inhibit the interaction of Ras-GTP with its effectors.Other recent reports identified small molecules that inhibitedSOS-mediated nucleotide exchange by binding to a pocket adjacent to theswitch I/II regions of KRas. Although compounds that exclusively inhibitGDP exchange for GTP may be effective in tumors that depend on wt Rasfor their malignancy, they are less likely to affect tumors with mtGTP-bound Ras.

Results

Assay 1:

The plant-GDP assay was used to identify compounds that inhibit theability of GTP to bind mt G12D KRas and to displace fluorescentlylabeled mant-GDP (29-deoxy-39-O—(N-methylanthraniloyl-GDP). Briefly, theassay consists of incubating purified mt KRas protein with want-GDP,with a reference fluorescence measurement taken when mant-GDP is boundto KRas. The addition of non-labeled GTP causes a binding competitionand displacement of mant-GDP from mt KRas, resulting in a loweredfluorescence signal. Compounds that prevent this decrease influorescence signal in the presence of non-labeled GTP may demonstrateallosteric inhibition of the ability of GTP to bind mt KRas.Optimization of this assay showed that both GTP and GDP were veryeffective at displacing mant-GDP from mt G12D KRas, with a slightpreference for GTP (FIG. 1A), as reported previously for mt G12C KRas.

Assay 2:

The GST-RBD ELISA assay was used to identify compounds that inhibit thebinding of mt KRas to the Ras binding domain (RBD) of its effectorRaf-1. This assay was adapted from an established ELISA assay. Briefly,the assay consists of first loading mt G12D KRas with 100 μM. GTP or GDPin TBS buffer (25 mM Tris, 0.15M NaCl, pH 7.4) and 10 mM EDTA, followedby 60 mM MgCl₂, then diluting mt KRas in binding buffer (2 mM MgCl₂, 10mM Na₂CO₃, 30 mM NaHCO₃, pH 9.6) prior to incubating in a 96-well plateovernight. After blocking and washing, GST-RBD was added (compounds canalso be added at this step), followed by HRP-conjugated anti-GSTantibody and then by TMB substrate and reading the absorbance at 450 nm.Optimization of this assay showed that GST-RBD binds with higheraffinity to GTP-versus GDP-bound mt G12D KRas (FIG. 1B).

Chemical Library:

The Torrey Pines Institute for Molecular Studies (TPIMS) library, amixture-based synthetic combinatorial library containing over 6 millioncompounds prepared as 62 mixtures varying in size between 1,000 and800,000 compounds per mixture, was screened. The 62 mixtures arereferred to as the “scaffold ranking library” since each mixture samplecontains only compounds with the same specific core scaffold with closestructural analogs. Hits identified from the scaffold ranking librarywere de-convoluted through subsequent screens of the correspondingpositional scanning libraries, and based on the data generated fromthese libraries, 20 to 50 individual compounds were selected,synthesized, and evaluated. This is a highly innovative approach to scana rich chemical space of over 6 million compounds by screening only 100sto 1000s of wells. The highly diverse and dense chemical space that theTPIMS library occupies coupled with its outstanding drug-like propertiesled to its extensive use to identify activity cliffs and unique leadsfor optimization for preclinical and clinical studies.

As shown in FIG. 2, the scaffold ranking library was used in themint-GDP assay to identify chemical scaffolds that inhibit the abilityof GTP to displace mant-GDP from mt G12D KRas. Focus was placed first onthe most potent 2239 mixture sample, which contains 1,008 compounds thatare all derived from the common imidazothiazole scaffold (FIG. 2).Screening of the positional scanning library for 2239, which consists of66 mixtures that are systematically formatted with structural analogs aswell as of selected individual compounds, led to several hits with themost potent, 87 (FIG. 3A), inhibiting the ability of GTP to displacewant-GDP from mt G12D KRas with IC₅₀ of 61 μM (FIG. 3B). A closelyrelated analogue, 92, was much less active (FIG. 3B), suggesting thatthe methyl group or 92 was helpful to the binding. GTP titration curvesshow that in the absence of compounds, the IC₅₀ of GTP to displacemant-GDP was 0.62 μM, whereas in the presence of 87, the IC₅₀ was13-fold higher at 7.2 μM (FIG. 3C). 92 had little effect on the abilityof GTP to displace mant-GDP (IC₅₀=0.79 μM).

In addition, other hits were identified from the 2239 positionalscanning library screens as well as from the original scaffold rankinglibrary such as the sample mixture 2137(dihydroimidazolyl-butyl-diketopiperazine scaffold) and sample mixture2291 (disubstituted-Aryl-thiazole-piperazine scaffold) (FIG. 2).

When a cutoff value of 60% inhibition was used, the structures of thesecompounds are listed in FIG. 6.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A compound having Formula I:

wherein R¹ is an alkyl group; R² is a phenyl group optionallysubstituted with amino, alkyl, alkenyl, alkynyl, alkoxyl, aryl,carboxylate, cyano, guanidinyl, halogen, haloalkyl, heteroaryl having2-8 carbon atoms and 1-3 heteroatoms selected from O, N and S, hydroxyl,nitrile, nitro, sulfinyl, sulfanyl, or mercapto; and R³ is H or an alkylgroup; or a pharmaceutical acceptable salt thereof.
 2. The compound ofclaim 1, wherein R³ is alkyl.
 3. The compound of claim 1, wherein R² isunsubstituted phenyl.
 4. The compound of claim 1, wherein the compoundhas Formula IA


5. The compound of claim 1, wherein the compound has Formula IB

wherein R⁵ is hydrogen, amino, alkyl, alkenyl, alkynyl, alkoxyl, aryl,carboxylate, cyano, guanidinyl, halogen, haloalkyl, heteroaryl having2-8 carbon atoms and 1-3 heteroatoms selected from O, N and S, hydroxyl,nitrile, nitro, sulfinyl, sulfanyl, or mercapto.
 6. The compound ofclaim 1, wherein R¹ is C₁₋₄ alkyl.
 7. The compound of claim 1, whereinthe compound is


8. The compound of claim 1, wherein the compound is


9. A pharmaceutical composition, comprising a therapeutically effectiveamount of a compound of claim 1 in a pharmaceutically acceptableexcipient.